UV excitable energy transfer reagents

ABSTRACT

Novel energy transfer dyes which can be used with shorter wavelength light sources are provided. These dyes include a donor dye with an absorption maxima at a wavelength between about 250 to 450 nm and an acceptor dye which is capable of absorbing energy emitted from the donor dye. One of the energy transfer dyes has a donor dye which is a member of a class of dyes having a coumarin or pyrene ring structure and an acceptor dye which is capable of absorbing energy emitted from the donor dye, wherein the donor dye has an absorption maxima between about 250 and 450 nm and the acceptor dye has an emission maxima at a wavelength greater than about 500 nm.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to fluorescent dyes and, morespecifically, energy transfer fluorescent dyes and their use.

2. Description of Related Art

A variety of fluorescent dyes have been developed for labeling anddetecting components in a sample. In general, fluorescent dyespreferably have a high quantum yield and a large extinction coefficientso that the dye may be used to detect small quantities of the componentbeing detected. Fluorescent dyes also preferably have a large Stokes'shift (i.e., the difference between the wavelength at which the dye hasmaximum absorbance and the wavelength at which the dye has maximumemission) so that the fluorescent emission is readily distinguished fromthe light source used to excite the dye.

One class of fluorescent dyes which has been developed is energytransfer fluorescent dyes. In general, energy transfer fluorescent dyesinclude a donor fluorophore and an acceptor fluorophore. In these dyes,when the donor and acceptor fluorophores are positioned in proximitywith each other and with the proper orientation relative to each other,the energy emission from the donor fluorophore is absorbed by theacceptor fluorophore and causes the acceptor fluorophore to fluoresce.It is therefore important that the excited donor fluorophore be able toefficiently absorb the excitation energy of the donor fluorophore andefficiently transfer the energy to the acceptor fluorophore.

A variety of energy transfer fluorescent dyes have been described in theliterature. For example, U.S. Pat. No. 4,996,143 and WO 95/21266describe energy transfer fluorescent dyes where the donor and acceptorfluorophores are linked by an oligonucleotide chain. Lee, et al.,Nucleic Acids Research 20:10 2471–2483 (1992) describes an energytransfer fluorescent dye which includes 5-carboxy rhodamine linked to4′-aminomethyl-5-carboxy fluorescein by the 4′-aminomethyl substituenton fluorescein. U.S. Pat. No. 5,847,162 describes additional classes ofenergy transfer dyes.

Several diagnostic and analytical assays have been developed whichinvolve the detection of multiple components in a sample usingfluorescent dyes, e.g. flow cytometry (Lanier, et al., J. Immunol. 132151–156 (1984)); chromosome analysis (Gray, et al., Chromosoma 73 9–27(1979)); and DNA sequencing. For these assays, it is desirable tosimultaneously employ a set of two or more spectrally resolvablefluorescent dyes so that more than one target substance can be detectedin the sample at the same time. Simultaneous detection of multiplecomponents in a sample using multiple dyes reduces the time required toserially detect individual components in a sample. In the case ofmulti-loci DNA probe assays, the use of multiple spectrally resolvablefluorescent dyes reduces the number of reaction tubes that are needed,thereby simplifying the experimental protocols and facilitating themanufacturing of application-specific kits. In the case of automated DNAsequencing, the use of multiple spectrally resolvable fluorescent dyesallows for the analysis of all four bases in a single lane therebyincreasing throughput over single-color methods and eliminatinguncertainties associated with inter-lane electrophoretic mobilityvariations. Connell, et al., Biotechniques 5 342–348 (1987); Prober, etal., Science 238 336–341 (1987), Smith, et al., Nature 321 674–679(1986); and Ansorge, et al., Nucleic Acids Research 15 4593–4602 (1989).

There are several difficulties associated with obtaining a set offluorescent dyes for simultaneously detecting multiple target substancesin a sample, particularly for analyses requiring an electrophoreticseparation and treatment with enzymes, e.g., DNA sequencing. Forexample, each dye in the set must be spectrally resolvable from theother dyes. It is difficult to find a collection of dyes whose emissionspectra are spectrally resolved, since the typical emission bandhalf-width for organic fluorescent dyes is about 40–80 nanometers (nm)and the width of the available spectrum is limited by the excitationlight source. As used herein the term “spectral resolution” in referenceto a set of dyes means that the fluorescent emission bands of the dyesare sufficiently distinct, i.e., sufficiently non-overlapping, thatreagents to which the respective dyes are attached, e.g.polynucleotides, can be distinguished on the basis of the fluorescentsignal generated by the respective dyes using standard photodetectionsystems, e.g. employing a system of band pass filters andphotomultiplier tubes, charged-coupled devices and spectrographs, or thelike, as exemplified by the systems described in U.S. Pat. Nos.4,230,558, 4,811,218, or in Wheeless et al, pgs. 21–76, in FlowCytometry: Instrumentation and Data Analysis (Academic Press, New York,1985).

The fluorescent signal of each of the dyes must also be sufficientlystrong so that each component can be detected with sufficientsensitivity. For example, in the case of DNA sequencing, increasedsample loading can not compensate for low fluorescence efficiencies,Pringle et al., DNA Core Facilities Newsletter, 1 15–21 (1988). Thefluorescent signal generated by a dye is generally greatest when the dyeis excited at its absorbance maximum. It is therefore preferred thateach dye be excited at about its absorbance maximum.

A further difficulty associated with the use of a set of dyes is thatthe dyes generally do not have the same absorbance maximum. When a setof dyes are used which do not have the same absorbance maximum, a tradeoff is created between the higher cost associated with providingmultiple light sources to excite each dye at its absorbance maximum, andthe lower sensitivity arising from each dye not being excited at itsabsorbance maximum.

In addition to the above difficulties, the charge, molecular size, andconformation of the dyes must not adversely affect the electrophoreticmobilities of the fragments. The fluorescent dyes must also becompatible with the chemistry used to create or manipulate thefragments, e.g., DNA synthesis solvents and reagents, buffers,polymerase enzymes, ligase enzymes, and the like.

Because of the multiple constraints on developing a set of dyes formulticolor applications, particularly in the area of four color DNAsequencing, only a few sets of fluorescent dyes have been developed.Connell, et al., Biotechniques 5 342–348 (1987); Prober, et al., Science238 336–341 (1987); and Smith, et al., Nature 321 674–679 (1986); andU.S. Pat. No. 5,847,162.

Energy transfer fluorescent dyes possess several features which makethem attractive for use in the simultaneous detection of multiple targetsubstances in a sample, such as in DNA sequencing. For example, a singledonor fluorophore can be used in a set of energy transfer fluorescentdyes so that each dye has strong absorption at a common wavelength.Then, by varying the acceptor fluorophore in the energy transfer dye, aseries of energy transfer dyes having spectrally resolvable fluorescenceemissions can be generated.

Energy transfer fluorescent dyes also provide a larger effective Stokes'shift than non-energy transfer fluorescent dyes. This is because theStokes' shift for an energy transfer fluorescent dye is based on thedifference between the wavelength at which the donor fluorophoremaximally absorbs light and the wavelength at which the acceptorfluorophore maximally emits light. In general, a need exists forfluorescent dyes having larger Stokes' shifts.

The sensitivity of any assay using a fluorescent dye is dependent on thestrength of the fluorescent signal generated by the fluorescent dye. Aneed therefore exists for fluorescent dyes which have a strongfluorescence signal. With regard to energy transfer fluorescent dyes,the fluorescence signal strength of these dyes is dependent on howefficiently the acceptor fluorophore absorbs the energy emission of thedonor fluorophore.

SUMMARY OF THE INVENTION

The present invention relates to energy transfer dyes which can be usedwith shorter wavelength light sources. The present invention alsorelates to reagents which include the energy transfer dyes of thepresent invention. The present invention also relates to methods whichuse dyes and reagents adapted to shorter wavelength light sources. Kitsare also provided which include the dyes and reagents.

Energy transfer dyes are provided which include a donor dye with anabsorption maxima at a wavelength between about 250 to 450 nm and anacceptor dye which is capable of absorbing energy from the donor dye.

It is noted that energy transfer may occur by a variety of mechanisms.For example, the emission of the donor dye does not need to overlap withthe absorbance of the acceptor dye for many of the dyes of the presentinvention.

In one variation, the donor dye has an absorption maxima between about300 and 450 nm, more preferably between about 350 and 400 nm.

The acceptor dye preferably has an emission maxima greater than about500 nm. In one variation, the acceptor dye has an emission maxima at awavelength greater than about 550 nm. The acceptor dye may also have anemission maxima at a wavelength between about 500 and 700 nm. Theacceptor dye may also be selected relative to the donor dye such thatthe acceptor dye has an emission maxima at a wavelength at least about150 nm greater than the absorption maxima of the donor dye.

In another embodiment of the present invention, the energy transfer dyehas a donor dye which is a member of a class of dyes having a coumarinor pyrene ring structure and an acceptor dye which is capable ofabsorbing energy from the donor dye.

In one variation of this embodiment, the donor dye has an absorptionmaxima between about 250 and 450 nm, preferably between about 300 and450 nm, and more preferably between about 350 and 400 nm.

In another variation of this embodiment, the acceptor dye has anemission maxima at a wavelength greater than about 500 nm, andoptionally more than 550 nm. The acceptor dye may also have an emissionmaxima at a wavelength between about 500 and 700 nm. The acceptor dyemay also be selected relative to the donor dye such that the acceptordye has an emission maxima at a wavelength at least about 150 nm greaterthan the absorption maxima of the donor dye.

An energy transfer dye according to the present invention may also havethe structure of “antennae” dyes or dendrimers in which large numbers ofdonor dyes are coupled to one acceptor dye where the donor dye eitherhas an absorption maxima between 250 and 450 nm or has a coumarin orpyrene ring structure.

The present invention also relates to fluorescent reagents containingany of the energy transfer dyes of the present invention. In general,these reagents include any molecule or material to which the energytransfer dyes of the invention can be attached. The presence of thereagent is detected by the fluorescence of the energy transfer dye. Oneuse of the reagents of the present invention is in nucleic acidsequencing.

Examples of classes of the fluorescent reagents include deoxynucleosidesand mono-, di- or triphosphates of a deoxynucleoside labeled with anenergy transfer dye. Examples of deoxynucleotides include deoxycytosine,deoxyadenosine, deoxyguanosine or deoxythymidine, and analogs andderivatives thereof.

Other classes of the reagents include analogs and derivatives ofdeoxynucleotides which are not extended at the 3′ position by apolymerase. A variety of analogs and derivatives have been developedwhich include a moiety at the 3′ position to prevent extension includinghalides, acetyl, benzyl and azide groups. Dideoxynucleosides anddideoxynucleoside mono-, di- or triphosphates which cannot be extendedhave also been developed. Examples of dideoxynucleotides includedideoxycytosine, dideoxyadenosine, dideoxyguanosine or dideoxythymidine,and analogs and derivatives thereof.

The fluorescently labeled reagent may also be an oligonucleotide. Theoligonucleotide may have a 3′ end which is extendable by using anucleotide polymerase. Such a labeled oligonucleotide may be used, forexample, as a dye-labeled primer in nucleic acid sequencing.

The present invention also relates to methods which use the energytransfer dyes and reagents of the present invention. In one embodiment,the method includes forming a series of different sized oligonucleotideslabeled with an energy transfer dye of the present invention, separatingthe series of labeled oligonucleotides based on size and detecting theseparated labeled oligonucleotides based on the fluorescence of theenergy transfer dye.

In another embodiment, the method includes forming a mixture of extendedlabeled primers by hybridizing a nucleic acid with an oligonucleotideprimer in the presence of deoxynucleoside triphosphates, at least onedideoxynucleoside triphosphate and a DNA polymerase, the DNA polymeraseextending the primer with the deoxynucleoside triphosphates until adideoxynucleoside triphosphate is incorporated which terminatesextension of the primer. Once terminated, the mixture of extendedprimers are separated and the separated extended primers detected bydetecting an energy transfer dye of the present invention that wasincorporated onto either the oligonucleotide primer, a deoxynucleotidetriphosphate, or a dideoxynuceotide triphosphate.

The present invention also relates to methods for sequencing a nucleicacid using the energy transfer dyes of the present invention. In oneembodiment, the method includes forming a mixture of extended labeledprimers by hybridizing a nucleic acid sequence with an oligonucleotideprimer in the presence of deoxynucleoside triphosphates, at least onedideoxynucleoside triphosphate and a DNA polymerase. The oligonucleotideprimer and/or the dideoxynucleotide is labeled with an energy transferdye of the present invention. The DNA polymerase is used to extend theprimer with the deoxynucleoside triphosphates until a dideoxynucleosidetriphosphate is incorporated which terminates extension of the primer.The mixture of extended primers are then separated and the sequence ofthe nucleic acid determined by detecting the energy transfer dye on theextended primer.

The present invention also relates to methods for detectingoligonucleotides and reagents labeled with energy transfer dyes usingshorter wavelength light sources. The light sources used in thesemethods preferably provide energy at a wavelength less than 450 nm. Inone variation, the light source provides energy at a wavelength betweenabout 250 and 450 nm, preferably between about 300 and 450 nm, and mostpreferably between about 350 and 450 nm. In one particular embodiment,the light source used provides energy at about 400 nm.

In one embodiment, the method includes forming a series of differentsized oligonucleotides labeled with an energy transfer dye, separatingthe series of labeled oligonucleotides based on size and detecting theseparated labeled oligonucleotides based on the fluorescence of theenergy transfer dye upon exposure to a shorter wavelength light source.

In another embodiment, the method includes forming a mixture of extendedlabeled primers by hybridizing a nucleic acid with an oligonucleotideprimer in the presence of deoxynucleoside triphosphates, at least onedideoxynucleoside triphosphate and a DNA polymerase, the DNA polymeraseextending the primer with the deoxynucleoside triphosphates until adideoxynucleoside triphosphate is incorporated which terminatesextension of the primer. Once terminated, the mixture of extendedprimers are separated. The separated extended primers are detected byexposing the extended primer to light having a wavelength between about250 and 450 nm and measuring light emitted by an energy transfer dye ata wavelength greater than about 500 nm. The energy transfer dye isincorporated onto either the oligonucleotide primer, a deoxynucleotidetriphosphate, or a dideoxynuceotide triphosphate.

The present invention also relates to methods for sequencing a nucleicacid using a shorter wavelength light source. In one embodiment, themethod includes forming a mixture of extended labeled primers byhybridizing a nucleic acid sequence with an oligonucleotide primer inthe presence of deoxynucleoside triphosphates, at least onedideoxynucleoside triphosphate and a DNA polymerase. The oligonucleotideprimer and/or the dideoxynucleotide is labeled with an energy transferdye adapted for use with a shorter wavelength light source. The DNApolymerase is used to extend the primer with the deoxynucleosidetriphosphates until a dideoxynucleoside triphosphate is incorporatedwhich terminates extension of the primer. The mixture of extendedprimers are then separated and the sequence of the nucleic aciddetermined by exposing the extended primer to light having a wavelengthbetween about 250 and 450 nm and measuring light emitted by the energytransfer dye at a wavelength greater than about 500 nm.

In a preferred variation of the embodiment, the extended primer isexposed to light having a wavelength between about 300 and 450 nm. Theextended primer may also be exposed to light having a wavelength betweenabout 350 and 400 nm. In another preferred variation of the embodiment,the light emitted by the energy transfer dye has a wavelength greaterthan about 550 nm. The light emitted by the energy transfer dye may alsohave a wavelength between about 500 and 700 nm. In another embodiment,the light emitted by the energy transfer dye has a wave length at leastabout 150 nm greater than the wavelength of the light to which theextended primer is exposed.

The present invention also relates to kits containing the dyes andreagents for performing DNA sequencing using the dyes and reagents ofthe present invention. A kit may include a set of 2, 3, 4 or more energytransfer dyes or reagents of the present invention. Optionally the kitsmay further include a nucleotide polymerase, additional nucleotidesand/or reagents useful for performing nucleic acid sequencing.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates examples of energy transfer dyes according to thepresent invention.

FIG. 2 illustrates examples of donor dyes which include a pyrene ringstructure.

FIG. 3 illustrates examples of donor dyes which include a coumarin ringstructure.

FIG. 4 illustrates the structure of a dendrimer energy-transfer dye.

FIG. 5 illustrates classes of acceptor dyes including xanthene dyes,cyanine dyes, phthalocyanine dyes and squaraine dyes.

FIG. 6 illustrates the general structure of xanthene dyes and classes ofxanthene dyes like fluorescein, rhodamine and asymmetric benzoxanthene.

FIG. 7 illustrates structures of acceptor dyes which may be used in thedyes of the present invention.

FIG. 8 illustrates examples of —C(O)R₂₂— subunits of linkers which maybe used in the present invention.

FIG. 9 illustrates the synthesis scheme of energy transfer dye DYE104.

FIG. 10 illustrates the synthesis scheme of energy transfer dye DYE106.

FIG. 11 illustrates the synthesis scheme of energy transfer dye DYE108.

FIG. 12 shows the fluorescence emission spectra of energy transfer dyesaccording to the present invention.

FIG. 13 illustrates the synthesis scheme of energy transfer dye DYE120.

FIG. 14 shows the fluorescence emission spectra of energy transfer dyeDYE120 according to the present invention.

DETAILED DESCRIPTION

The present invention relates to energy transfer dyes which may be usedwith shorter wavelength light sources. For example, the energy transferdyes are preferably adapted to be excited at wavelengths between about250 and 450 nm. The present invention also relates to reagents whichinclude the energy transfer dyes of the present invention. The presentinvention further relates to methods which use the dyes and reagents.Kits are also provided which include the dyes and reagents.

I. Energy Transfer Dyes

The energy transfer dyes of the present invention include a donor dyeand an acceptor dye which is capable of emitting energy in response toabsorbing energy from the donor dye.

In one embodiment, the energy transfer dyes may be excited atwavelengths between about 250 and 450 nm. According to this embodiment,the donor dye preferably has an absorption maxima at a wavelengthbetween about 250 to 450 nm, more preferably between about 300 and 450nm, and most preferably between about 350 and 450 nm.

In another embodiment, the energy transfer dyes include an donor dyehaving a coumarin or pyrene ring structure.

The acceptor dye may be any dye which is capable of absorbing energyfrom the donor dye. In one embodiment, the acceptor dye has an emissionmaxima greater than about 500 nm, more preferably greater than 550 nm.In another embodiment, the acceptor dye has an emission maxima betweenabout 500 and 700 nm. In another embodiment, the acceptor dye isselected such that it has an emission maxima at a wavelength at leastabout 150 nm greater than the absorption maxima of the donor dye.

The energy transfer dyes may also include a linker which couples thedonor dye to the acceptor dye. The linker preferably couples the donordye to the acceptor dye such that the acceptor dye is able to absorbsubstantially all of the energy by the donor dye.

Particular examples of energy transfer dyes of the present invention areillustrated in FIG. 1. In these examples 5-carboxyfluorescein, which hasan emission maxima of 523 nm, is used as the acceptor dye.Coumarin-based donor dyes DYE116, which has an absorption maxima at 376nm, DYE114 (absorption maximum=328 nm), and DYE112 (absorptionmaximum=362 nm) or pyrene-based donor dye DYE110 (absorption maximum=396nm) are conjugated to a 5-carboxyfluorescein acceptor derivatized with a4-aminomethylbenzoic linker (5CF-B). The structures of the 5CF-Bconjugates, DYE102, DYE104, DYE106, and DYE108, are shown in FIG. 1.

A. Donor Dye

In one embodiment, the donor dye has an absorption maxima at awavelength between about 250 to 450 nm, more preferably between about300 and 450 nm, most preferably between about 350 and 400 nm.

In another embodiment, the donor dye has a pyrene ring structure. Asused herein, pyrene dyes include all molecules including the generalstructure

The present invention is intended to encompass all pyrene dyes since allmay be used in the present invention. Particular examples of pyrenedyes, DYE110, DYE122, DYE124 and DYE126, are illustrated in FIG. 2. Inthe figure, X is a functional group which may be used to attachsubstituents, such as the acceptor dye, to the donor dye.

In another embodiment, the donor dye has a coumarin ring structure. Asused herein, coumarin dyes include all molecules including the generalstructure.

The present invention is intended to encompass all coumarin dyes sinceall may be used in the present invention. Particular examples ofcoumarin dyes are illustrated in FIG. 3. In the figure, X is afunctional group which may be used to attach substituents, such as theacceptor dye, to the donor dye.

The present invention also relates to energy transfer dyes wheremultiple donor dyes are coupled to an acceptor dye. Coumarin dyes arewater-soluble and coumarin conjugates show much better quantum yieldsthan larger dyes, for which the quantum yields in water are about ⅓ thatof free acceptor dyes. The present invention utilizes the small size andsolubility of the coumarins to synthesize “antennae” dyes or dendrimersin which large numbers of donor dyes are coupled to one acceptor dye. Anexample of a dendrimer energy transfer dye (DYE118) is shown in FIG. 4.

B. Acceptor Dye

The acceptor dye may be any dye which is capable of absorbing energyfrom the donor dye. In one embodiment, the acceptor dye has an emissionmaxima greater than about 500 nm, more preferably greater than 550 nm.In another embodiment, the acceptor dye has an emission maxima betweenabout 500 and 700 nm. In another embodiment, the acceptor dye isselected such that it has an emission maxima at a wavelength at leastabout 150 nm greater than the absorption maxima of the donor dye.

Examples of classes of acceptor dyes which may be used in the energytransfer fluorescent dye of this embodiment include, but are not limitedto, xanthene dyes, cyanine dyes, phthalocyanine dyes and squaraine dyes.The general structures of these dyes are illustrated in FIG. 5. Thesubstituents illustrated on these dyes may be selected from the widevariety of substituents which may be incorporated onto these differentclasses of dyes since all dyes having the general xanthene, fluorescein,rhodamine, asymmetric benzoxanthene, cyanine, phthalocyanine andsquaraine ring structures are intended to fall within the scope of thisinvention.

One particular class of acceptor dyes which may be used in the energytransfer dyes of the present invention are xanthene dyes. As usedherein, xanthene dyes include all molecules having the general structureillustrated in FIG. 6 where Y₁ and Y₂ taken separately are eitherhydroxyl, oxygen, iminium or amine, the iminium and amine preferablybeing a tertiary iminium or amine. Examples of classes of xanthene dyesare fluorescein, rhodamine and asymmetric benzoxanthene classes of dyeswhich are also illustrated in FIG. 6. The substituents illustrated onthese dyes may be selected from the wide variety of substituents whichmay be incorporated onto these different classes of dyes since all dyeshaving the general xanthene, fluorescein, rhodamine, and asymmetricbenzoxanthene ring structures are intended to fall within the scope ofthis invention. Fluorescein and rhodamine dyes may be linked to asubstituent, such as an acceptor dye, a nucleoside, or anoligonucleotide, in a variety of locations. Illustrated with an asterik“*” in FIG. 6 are preferred locations for substitutions.

Fluorescein and rhodamine classes of dyes are members of a particularsubclass of xanthene dyes where R₁₇ is a phenyl or substituted phenylhaving the general formula

Substituents X₁–X₅ on the phenyl ring can include hydrogen, fluorine,chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate,amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents aretaken together to form a ring, and combinations thereof. As illustratedin FIG. 5, dyes where Y₁ is hydroxyl and Y₂ is carboxyl are fluoresceindyes and where Y₁ is amine and Y₂ is iminium are rhodamine dyes.

R₁₁–R₁₇ may be any substituent which is compatible with the energytransfer dyes of the present invention, it being noted that the R₁₁–R₁₇may be widely varied in order to alter the spectral and mobilityproperties of the dyes. Examples of R₁₁–R₁₇ substituents include, butnot limited to hydrogen, fluorine, chlorine, bromine, iodine, carboxyl,alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile,alkoxy, phenyl, substituted phenyl, where adjacent substituents aretaken together to form a ring, and combinations thereof.

In one embodiment, R₁₅ and R₁₆ are taken together to form a substitutedor unsubstituted benzene ring. This class of xanthene dyes are referredto herein as asymmetric benzoxanthene dyes and are described in U.S.Pat. No. 5,840,999, entitled Asymmetric Benzoxanthene Dyes, by Scott C.Benson, et al. which is incorporated herein by reference.

In one particular embodiment, the acceptor dye is a member of the classof dyes where Y₁ is amine, Y₂ is iminium, and X₂ and X₅ are chlorine,referred to herein as 4,7-dichlororhodamine dyes. Dyes falling withinthe 4,7-dichlororhodamine class of dyes and their synthesis aredescribed in U.S. Pat. No. 5,847,162, entitled: “4,7-DichlororhodamineDyes” which is incorporated herein by reference.

R₁₁–R₁₇ and X₁–X₅ may also each independently be a linking moiety whichmay be used to attach the energy transfer dye to a reagent, such as anucleotide, nucleoside or oligonucleotide. Examples of linking moietiesinclude isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinylamine,succinimidyl ester, or other active carboxylate whenever thecomplementary functionality is amine. Preferably the linking group ismaleimide, halo acetyl, or iodoacetamide whenever the complementaryfunctionality is sulfhydryl. See R. Haugland, Molecular Probes Handbookof Fluorescent Probes and Research Chemicals, Molecular probes, Inc.(1992). In a particularly preferred embodiment, the linking group is anactivated NHS ester formed from a carboxyl group on either the donor oracceptor dye which can be reacted with an aminohexyl-oligomer to form adye labeled oligonucleotide primer.

As used here, alkyl denotes straight-chain and branched hydrocarbonmoieties, i.e., methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl,sec-butyl, neopentyl, tert-pentyl, and the like. Substituted alkyldenotes an alkyl moiety substituted with any one of a variety ofsubstituents, including, but not limited to hydroxy, amino, thio, cyano,nitro, sulfo, and the like. Haloalkyl denotes a substituted alkyl withone or more halogen atom substituents, usually fluoro, chloro, bromo, oriodo. Alkene denotes a hydocarbon wherein one or more of thecarbon-carbon bonds are double bonds, and the non-double bonded carbonsare alkyl or substituted alkyl. Alkyne denotes a hydocarbon where one ormore of the carbons are bonded with a triple bond and where thenon-triple bonded carbons are alkyl or substituted alkyl moieties.Sulfonate refers to moieties including a sulfur atom bonded to 3 oxygenatoms, including mono- and di-salts thereof, e.g., sodium sulfonate,potassium sulfonate, disodium sulfonate, and the like. Amino refers tomoieties including a nitrogen atom bonded to 2 hydrogen atoms, alkylmoieties, or any combination thereof. Amido refers to moieties includinga carbon atom double bonded to an oxygen atom and single bonded to anamino moiety. Nitrile refers to moieties including a carbon atom triplebonded to a nitrogen atom. Alkoxy refers to a moiety including an alkylmoiety single bonded to an oxygen atom. Aryl refers to single ormultiple phenyl or substituted phenyl, e.g., benzene, naphthalene,anthracene, biphenyl, and the like.

In another embodiment, the acceptor dye is selected such that theacceptor dye has an emission maximum that is greater than about 500 nmand an emission maximum that is at least about 150 nm greater than theabsorption maxima of the donor dye. This class of dyes of the presentinvention exhibit unusually large Stokes' shifts, as measured by thedifference between the absorbance of the donor and the emission of theacceptor. In addition, these dyes exhibit efficient energy transfer inthat minimal donor fluorescence is observed. Interestingly, energy istransferred from the donor to the acceptor in some of the dyes belongingto this class even though the absorbance spectrum of the acceptor dyedoes not overlap with the emission spectrum of the donor dye.

Particular examples of acceptor dyes which may be used in the dyes ofthe present invention include, but are not limited to isomers ofcarboxyfluorescein (e.g., 5 and 6 carboxy), 4,7-dichlorofluoresceins,4,7-dichlororhodamines, fluoresceins, asymmetric benzoxanthene dyes,isomers of carboxy-HEX (e.g., 5 and 6 carboxy), NAN, CI-FLAN, TET, JOE,ZOE, rhodamine, isomers of carboxyrhodamine (e.g., 5 and 6 carboxy),isomers of carboxy R110 (e.g., 5 and 6 carboxy), isomers of carboxy R6G(e.g., 5 and 6 carboxy), 4,7-dichlorofluoresceins (See U.S. Pat. No.5,188,934), 4,7-dichlororhodamines (See U.S. Pat. No. 5,847,162),asymmetric benzoxanthene dyes (See U.S. Pat. No. 5,840,999), isomers ofN,N,N′,N′-tetramethyl carboxyrhodamine (TAMRA) (e.g., 5 and 6 carboxy),isomers of carboxy-X-rhodamine (ROX) (e.g., 5 and 6 carboxy) and Cy5.Illustrated in FIG. 7 are the structures of these dyes.

C. Linkers

The donor dye may be joined with the acceptor dye using a wide varietyof linkers which have been developed, all of which are intended to fallwithin the scope of the present invention. The energy transfer dyeswhich include a linker may generally be illustrated as

Donor ---- Linker ---- Acceptor

In a preferred embodiment, the linker joins the donor dye to theacceptor dye such that the acceptor dye absorbs substantially all of theenergy by the donor dye. While not being bound by theory, it is believedthat the efficiency of energy transmission from the donor dye to theacceptor dye is dependent upon the separation between the dyes andrelative orientation of the dyes. Described in U.S. Pat. No. 5,800,996are linkers which have been found to be effective for providing a veryhigh level of energy transfer between the donor and acceptor dye. U.S.Pat. No. 5,800,996 also describes methods for synthesizing dyesincorporating these linkers. U.S. Pat. No. 5,800,996 is incorporatedherein by reference in its entirety.

In one particular embodiment, the linker used in the energy transferdyes of the present invention is such that the acceptor dye absorbssubstantially all of the excitation energy by the donor dye. Suchlinkers may include a functional group which provides structuralrigidity to the linker. Examples of such functional groups include analkene, diene, alkyne, a five and six membered ring having at least oneunsaturated bond and/or having a fused ring structure.

Examples of functional groups with a five or six membered ring with atleast one unsaturatd bond and/or a fused ring structure includecyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan,thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran,pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine,indene, benzofuran, thionaphthene, indole and naphthalene.

One linker according to the present invention for linking a donor dye toan acceptor dye in an energy transfer dye includes the subunit structure—C(O)R₂₂—, where R₂₂ includes a functional group such as the onesdescribed above which provides structural rigidity. FIG. 8 illustratesexamples of —C(O)R₂₂— subunits of linkers which may be used in thelinkers of the present invention.

One embodiment of this linker has the general structure—R₂₁Z₁C(O)R₂₂R₂₈—, where R₂₁ is a C₁₋₅ alkyl attached to the donor dye,C(O) is a carbonyl group, Z₁ is either NH, sulfur or oxygen, R₂₂ is asubstituent which includes an alkene, diene, alkyne, a five and sixmembered ring having at least one unsaturated bond or a fused ringstructure which is attached to the carbonyl carbon, and R₂₈ includes afunctional group which attaches the linker to the acceptor dye.

In one embodiment of this linker, the linker has the general structure—R₂₁Z₁C(O)R₂₂R₂₉Z₂C(O)— where R₂₁ and R₂₂ are as detailed above, Z₁ andZ₂ are each independently either NH, sulfur or oxygen, R₂₉ is a C₁₋₅alkyl, and the terminal carbonyl group is attached to the ring structureof the acceptor dye. In the variation where Z₂ is nitrogen, the—C(O)R₂₂R₂₉Z₂— subunit forms an amino acid subunit.

A preferred embodiment of this linker is where R₂₁ and R₂₉ aremethylene, Z₁ and Z₂ are NH, and R₂₂ is benzene.

In yet another variation, the linker has the general formula R₂₅Z₃C(O)or R₂₅Z₃C(O)R₂₆Z₄C(O) where R₂₅ is attached to the donor dye, C(O) is acarbonyl group and the terminal carbonyl group is attached to theacceptor dye, R₂₅ and R₂₆ are each selected from the group of C₁₋₄alkyl, and Z₃ and Z₄ are each independently either NH, O or S.

In another variation of this embodiment, the linker includes a R₂₇Z₅C(O)group where R₂₇ is a C₁₋₅ alkyl attached to the donor dye, Z₅ is eitherNH, sulfur or oxygen, and C(O) is a carbonyl group attached to theacceptor dye.

II. Reagents Including Energy Transfer Dyes of the Present Invention

The present invention also relates to reagents which incorporate anenergy transfer dye according to the present invention. As described ingreater detail in Section III, these reagents may be used in a widevariety of methods for detecting the presence of a component in asample.

The reagents of the present invention include any molecule or materialto which the energy transfer dyes of the invention can be attached andused to detect the presence of the reagent based on the fluorescence ofthe energy transfer dye. Types of molecules and materials to which thedyes of the present invention may be attached to form a reagent include,but are not limited to proteins, polypeptides, polysaccharides,nucleotides, nucleosides, oligonucleotides, oligonucleotide analogs(such as a peptide nucleic acid), lipids, solid supports, organic andinorganic polymers, and combinations and assemblages thereof, such aschromosomes, nuclei, living cells, such as bacteria, othermicroorganisms, mammalian cells, and tissues.

Preferred classes of reagents of the present invention are nucleotides,nucleosides, oligonucleotides and oligonucleotide analogs which havebeen modified to include an energy transfer dye of the invention.Examples of uses for nucleotide and nucleoside reagents include, but arenot limited to, labeling oligonucleotides formed by enzymatic synthesis,e.g., nucleoside triphosphates used in the context of PCR amplification,Sanger-type nucleotide sequencing, and nick-translation reactions.Examples of uses for oligonucleotide reagents include, but are notlimited to, as DNA sequencing primers, PCR primers, oligonucleotidehybridization probes, and the like.

One particular embodiment of the reagents are labeled nucleosides, suchas cytosine, adenosine, guanosine, and thymidine, labeled with an energytransfer fluorescent dye of the present invention. These reagents may beused in a wide variety of methods involving oligonucleotide synthesis.Another related embodiment are labeled nucleotides (NTP), e.g., mono-,di- and triphosphate nucleoside phosphate esters. These reagentsinclude, in particular, deoxynucleoside triphosphates (dNTP), such asdeoxycytosine triphosphate, deoxyadenosine triphosphate, deoxyguanosinetriphosphate, and deoxythymidine triphosphate, labeled with an energytransfer fluorescent dye of the present invention. These reagents may beused, for example, as polymerase substrates in the preparation of dyelabeled oligonucleotides. These reagents also include labeleddideoxynucleoside triphosphates (ddNTP), such as dideoxycytosinetriphosphate, dideoxyadenosine triphosphate, dideoxyguanosinetriphosphate, and dideoxythymidine triphosphate, labeled with an energytransfer fluorescent dye of the present invention. These reagents may beused, for example, in dye termination sequencing.

Another embodiment of reagents are oligonucleotides which includes anenergy transfer dye of the present invention. These reagents may beused, for example, in dye primer sequencing.

As used herein, “nucleoside” refers to a compound consisting of apurine, deazapurine, or pyrimidine nucleoside base, e.g., adenine,guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, andthe like, linked to a pentose at the 1′ position, including 2′-deoxy and2′-hydroxyl forms, e.g. as described in Kornberg and Baker, DNAReplication, 2nd Ed. (Freeman, San Francisco, 1992). The term“nucleotide” as used herein refers to a phosphate ester of a nucleoside,e.g., mono, di and triphosphate esters, wherein the most common site ofesterification is the hydroxyl group attached to the C-5 position of thepentose. “Analogs” in reference to nucleosides include syntheticnucleosides having modified base moieties and/or modified sugarmoieties, e.g. described generally by Scheit, Nucleotide Analogs (JohnWiley, New York, 1980). The terms “labeled nucleoside” and “labelednucleotide” refer to nucleosides and nucleotides which are covalentlyattached to an energy transfer dye through a linkage.

As used herein, the term “oligonucleotide” refers to linear polymers ofnatural or modified nucleoside monomers, including double and singlestranded deoxyribonucleosides, ribonucleosides, α-anomeric formsthereof, and the like. Usually the nucleoside monomers are linked byphosphodiester linkages, where as used herein, the term “phosphodiesterlinkage” refers to phosphodiester bonds or analogs thereof includingphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate,phosphoramidate, and the like, including associated counterions, e.g.,H, NH₄, Na, and the like if such counterions are present. Theoligonucleotides range in size form a few monomeric units, e.g. 8–40, toseveral thousands of monomeric units. Whenever an oligonucleotide isrepresented by a sequence of letters, such as “ATGCCTG,” it will beunderstood that the nucleotides are in 5′→3′ order from left to rightand that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G”denotes deoxyguanosine, and “T” denotes thymidine, unless otherwisenoted.

Nucleoside labeling can be accomplished using any of a large number ofknown nucleoside labeling techniques using known linkages, linkinggroups, and associated complementary functionalities. The linkagelinking the dye and nucleoside should (i) be stable to oligonucleotidesynthesis conditions, (ii) not interfere with oligonucleotide-targethybridization, (iii) be compatible with relevant enzymes, e.g.,polymerases, ligases, and the like, and (iv) not quench the fluorescenceof the dye.

Preferably, the dyes are covalently linked to the 5-carbon of pyrimidinebases and to the 7-carbon of 7-deazapurine bases. Several suitable baselabeling procedures have been reported that can be used with theinvention, e.g. Gibson et al, Nucleic Acids Research, 15 6455–6467(1987); Gebeyehu et al, Nucleic Acids Research, 15 4513–4535 (1987);Haralambidis et al, Nucleic Acids Research, 15 4856–4876 (1987); Nelsonet al., Nucleosides and Nucleotides, 5(3) 233–241 (1986); Bergstrom, etal., JACS, 111 374–375 (1989); U.S. Pat. Nos. 4,855,225, 5,231,191, and5,449,767, each of which is incorporated herein by reference.

Preferably, the linkages are acetylenic amido or alkenic amido linkages,the linkage between the dye and the nucleotide base being formed byreacting an activated N-hydroxysuccinimide (NHS) ester of the dye withan alkynylamino-, alkynylethoxyamino- or alkenylamino-derivatized baseof a nucleotide. More preferably, the resulting linkage isproargyl-1-ethoxyamido (3-(amino)ethoxy-1-propynyl),3-(carboxy)amino-1-propynyl or 3-amino-1-propyn-1-yl.

Several preferred linkages for linking the dyes of the invention to anucleoside base are shown below.

where R₁ and R₂ taken separately are H, alkyl, a protecting group or afluorescent dye.

The synthesis of alkynylamino-derivatized nucleosides is taught by Hobbset al. in European Patent Application No. 87305844.0, and Hobbs et al.,J. Org. Chem., 54 3420 (1989), which is incorporated herein byreference. Briefly, the alkynylamino-derivatized nucleotides are formedby placing the appropriate halodideoxynucleoside (usually5-iodopyrimidine and 7-iodo-7-deazapurine dideoxynucleosides as taughtby Hobbs et al. (cited above)) and Cu(I) in a flask, flushing with argonto remove air, adding dry DMF, followed by addition of an alkynylamine,triethyl-amine and Pd(0). The reaction mixture can be stirred forseveral hours, or until thin layer chromatography indicates consumptionof the halodideoxynucleoside. When an unprotected alkynylamine is used,the alkynylamino-nucleoside can be isolated by concentrating thereaction mixture and chromatographing on silica gel using an elutingsolvent which contains ammonium hydroxide to neutralize the hydrohalidegenerated in the coupling reaction. When a protected alkynylamine isused, methanol/methylene chloride can be added to the reaction mixture,followed by the bicarbonate form of a strongly basic anion exchangeresin. The slurry can then be stirred for about 45 minutes, filtered,and the resin rinsed with additional methanol/methylene chloride. Thecombined filtrates can be concentrated and purified byflash-chromatography on silica gel using a methanol-methylene chloridegradient. The triphosphates are obtained by standard techniques.

The synthesis of oligonucleotides labeled with an energy transfer dye ofthe present invention can be accomplished using any of a large number ofknown oligonucleotide labeling techniques using known linkages, linkinggroups, and associated complementary functionalities. For example,labeled oligonucleotides may be synthesized enzymatically, e.g., using aDNA polymerase or ligase, e.g., Stryer, Biochemistry, Chapter 24, W. H.Freeman and Company (1981), or by chemical synthesis, e.g., by aphosphoramidite method, a phosphite-triester method, and the like, e.g.,Gait, Oligonucleotide Synthesis, IRL Press (1990). Labels may beintroduced during enzymatic synthesis utilizing labeled nucleosidetriphosphate monomers, or introduced during chemical synthesis usinglabeled non-nucleotide or nucleotide phosphoramidites, or may beintroduced subsequent to synthesis.

Generally, if the labeled oligonucleotide is made using enzymaticsynthesis, the following procedure may be used. A template DNA isdenatured and an oligonucleotide primer is annealed to the template DNA.A mixture of deoxynucleoside triphosphates is added to the reactionincluding dGTP, dATP, dCTP, and dTTP where at least a fraction of one ofthe deoxynucleotides is labeled with a dye compound of the invention asdescribed above. Next, a polymerase enzyme is added under conditionswhere the polymerase enzyme is active. A labeled polynucleotide isformed by the incorporation of the labeled deoxynucleotides duringpolymerase strand synthesis. In an alternative enzymatic synthesismethod, two primers are used instead of one, one primer complementary tothe + strand and the other complementary to the − strand of the target,the polymerase is a thermostable polymerase, and the reactiontemperature is cycled between a denaturation temperature and anextension temperature, thereby exponentially synthesizing a labeledcomplement to the target sequence by PCR, e.g., PCR Protocols, Innis etal. eds., Academic Press (1990).

Generally, if the labeled oligonucleotide is made using a chemicalsynthesis, it is preferred that a phosphoramidite method be used.Phosphoramidite compounds and the phosphoramidite method ofpolynucleotide synthesis are preferred in synthesizing oligonucleotidesbecause of the efficient and rapid coupling and the stability of thestarting materials. The synthesis is performed with the growingoligonucleotide chain attached to a solid support, so that excessreagents, which are in the liquid phase, can be easily removed byfiltration, thereby eliminating the need for purification steps betweencycles.

In view of the utility of phosphoramidite reagents in labelingnucleosides and oligonucleotides, the present invention also relates tophosphoramidite compounds which include an energy transfer dye of thepresent invention.

Nucleoside labeling with the dyes can be accomplished using any of alarge number of known nucleoside labeling techniques using knownlinkages, linking groups, and associated complementary functionalities.Preferably, the dyes are covalently linked to the 5-carbon of pyrimidinebases and to the 7-carbon of 7-deazapurine bases. Several suitable baselabeling procedures have been reported that can be used with theinvention, e.g. Gibson et al. Nucleic Acid Res. 15 6455–6467 (1987);Gebeyehu et al. Nucleic Acid Res. 15 4513–4535 (1987); Haralambidis etal. Nucleic Acid Res. 15 4856–4876; Nelson et al. Nucleosides andNucleotides 5 233–241 (1986); Bergstrom et al. J. Am. Chem. Soc.111:374–375 (1989); U.S. Pat. Nos. 4,855,225, 5,231,191, and 5,449,767,each of which is incorporated herein by reference.

Preferably, the linkages are acetylenic amido or alkenic amido linkages,the linkage between the dye and nucleotide base being formed by reactingan activated N-hydroxysuccinimide (NHS) ester of the dye with analkynylamino, alkynylethoxyamino, or alkenylamino-derivatized base of anucelotide. More preferably, the resulting linkage isproargyl-1-ethoxyamido (3-(amino)ethoxy-1-propynyl),3-(carboxy)amino-1-propynyl or 3-amino-1-propyn-1-yl.

The synthesis of alkynylamino-derivatized nucleosides is taught by Hobbset al. J. Org. Chem. 54:3420 (1989), which is incorporated herein byreference. Briefly, the alkynylamino-derivatized nucleosides are formedby placing the appropriate halodideoxynucleoside (usually5-iodopryrimidine and 7-iodo-deazapurine dideoxynucleosides as taught byHobbs et al. as cited above) and Cu(I) in a flask, flushing with argonto remove air, adding dry DMF, followed by addition of an alkynylamine,triethyl-amine and Pd(0). The reaction mixture can be stirred forseveral hours, or until thin layer chromatography indicates consumptionof the halodideoxynucleoside. When an unprotected alkynylamine is used,the alkynylaminonucleoside can be isolated by concentrating the reactionmixture and chromatographing on silica gel using an eluting solventwhich contains ammonium hydroxide to neutralize the hydrohalidegenerated in the coupling reaction. When a protected alkynylamine isused, methanol/methylene chloride can be added to the reaction mixture,followed by the bicarbonate form of a strongly basic anion exchangeresin. The slurry can then be stirred for about 45 minutes, filtered,and the resin rinsed with additional methanol/methylene chloride. Thecombined filtrate can be concentrated and purified byflash-chromatography on silica gel using a methanol-methylene chloridegradient. The triphosphates are obtained by standard techniques.

Detailed descriptions of the chemistry used to form oligonucleotides bythe phosphoramidite method are provided in Caruthers et al., U.S. Pat.No. 4,458,066; Caruthers et al., U.S. Pat. No. 4,415,732; Caruthers etal., Genetic Engineering, 4 1–17 (1982); Users Manual Model 392 and 394Polynucleotide Synthesizers, pages 6–1 through 6–22, Applied Biosystems,Part No. 901237 (1991), each of which are incorporated by reference intheir entirety.

The following briefly describes the steps of a typical oligonucleotidesynthesis cycle using the phosphoramidite method. First, a solid supportincluding a protected nucleotide monomer is treated with acid, e.g.,trichloroacetic acid, to remove a 5′-hydroxyl protecting group, freeingthe hydroxyl for a subsequent coupling reaction. An activatedintermediate is then formed by simultaneously adding a protectedphosphoramidite nucleoside monomer and a weak acid, e.g., tetrazole, tothe reaction. The weak acid protonates the nitrogen of thephosphoramidite forming a reactive intermediate. Nucleoside addition iscomplete within 30 s. Next, a capping step is performed which terminatesany polynucleotide chains that did not undergo nucleoside addition.Capping is preferably done with acetic anhydride and 1-methylimidazole.The internucleotide linkage is then converted from the phosphite to themore stable phosphotriester by oxidation using iodine as the preferredoxidizing agent and water as the oxygen donor. After oxidation, thehydroxyl protecting group is removed with a protic acid, e.g.,trichloroacetic acid or dichloroacetic acid, and the cycle is repeateduntil chain elongation is complete. After synthesis, the polynucleotidechain is cleaved from the support using a base, e.g., ammonium hydroxideor t-butyl amine. The cleavage reaction also removes any phosphateprotecting groups, e.g., cyanoethyl. Finally, the protecting groups onthe exocyclic amines of the bases and the hydroxyl protecting groups onthe dyes are removed by treating the polynucleotide solution in base atan elevated temperature, e.g., 55° C.

Any of the phosphoramidite nucleoside monomers may be dye-labeledphosphoramidites. If the 5′-terminal position of the nucleotide islabeled, a labeled non-nucleotidic phosphoramidite of the invention maybe used during the final condensation step. If an internal position ofthe oligonucleotide is to be labeled, a labeled nucleotidicphosphoramidite of the invention may be used during any of thecondensation steps. Subsequent to their synthesis, oligonucleotides maybe labeled at a number of positions including the 5′-terminus. SeeOligonucleotides and Analogs, Eckstein ed., Chapter 8, IRL Press (1991)and Orgel et al., Nucleic Acids Research 11(18) 6513 (1983); U.S. Pat.No. 5,118,800, each of which are incorporated by reference

Oligonucleotides may also be labeled on their phosphodiester backbone(Oligonucleotides and Analogs, Eckstein ed., Chapter 9) or at the3′-terminus (Nelson, Nucleic Acids Research 20(23) 6253–6259, and U.S.Pat. Nos. 5,401,837 and 5,141,813, both patents hereby incorporated byreference. For a review of oligonucleotide labeling procedures see R.Haugland in Excited States of Biopolymers, Steiner ed., Plenum Press, NY(1983).

In one preferred post-synthesis chemical labeling method anoligonucleotide is labeled as follows. A dye including a carboxy linkinggroup is converted to the n-hydroxysuccinimide ester by reacting withapproximately 1 equivalent of 1,3-dicyclohexylcarbodiimide andapproximately 3 equivalents of n-hydroxysuccinimide in dry ethyl acetatefor 3 hours at room temperature. The reaction mixture is washed with 5%HCl, dried over magnesium sulfate, filtered, and concentrated to a solidwhich is resuspended in DMSO. The DMSO dye stock is then added in excess(10–20×) to an aminohexyl derivatized oligonucleotide in 0.25 Mbicarbonate/carbonate buffer at pH 9.4 and allowed to react for 6 hours,e.g., U.S. Pat. No. 4,757,141. The dye labeled oligonucleotide isseparated from unreacted dye by passage through a size-exclusionchromatography column eluting with buffer, e.g., 0.1 molar triethylamineacetate (TEAA). The fraction containing the crude labeledoligonucleotide is further purified by reverse phase HPLC employinggradient elution.

III. Methods Employing Dyes and Reagents of the Present Invention

The energy transfer dyes and reagents of the present invention may beused in a wide variety of methods for detecting the presence of acomponent in a sample by labeling the component in the sample with areagent containing the dye. In particular, the energy transfer dyes andreagents of the present invention are well suited for use in methodswhich combine separation and fluorescent detection techniques,particularly methods requiring the simultaneous detection of multiplespatially-overlapping analytes. For example, the dyes and reagents areparticularly well suited for identifying classes of oligonucleotidesthat have been subjected to a biochemical separation procedure, such aselectrophoresis, where a series of bands or spots of target substanceshaving similar physiochemical properties, e.g. size, conformation,charge, hydrophobicity, or the like, are present in a linear or planararrangement. As used herein, the term “bands” includes any spatialgrouping or aggregation of analytes on the basis of similar or identicalphysiochemical properties. Usually bands arise in the separation ofdye-oligonucleotide conjugates by electrophoresis.

Classes of oligonucleotides can arise in a variety of contexts. In apreferred category of methods referred to herein as “fragment analysis”or “genetic analysis” methods, labeled oligonucleotide fragments aregenerated through template-directed enzymatic synthesis using labeledprimers or nucleotides, e.g., by ligation or polymerase-directed primerextension; the fragments are subjected to a size-dependent separationprocess, e.g., electrophoresis or chromatography; and, the separatedfragments are detected subsequent to the separation, e.g., bylaser-induced fluorescence. In a particularly preferred embodiment,multiple classes of oligonucleotides are separated simultaneously andthe different classes are distinguished by spectrally resolvable labels.

One such fragment analysis method is amplified fragment lengthpolymorphisim detection (AmpFLP) and is based on amplified fragmentlength polymorphisms, i.e., restriction fragment length polymorphismsthat are amplified by PCR. These amplified fragments of varying sizeserve as linked markers for following mutant genes through families. Thecloser the amplified fragment is to the mutant gene on the chromosome,the higher the linkage correlation. Because genes for many geneticdisorders have not been identified, these linkage markers serve to helpevaluate disease risk or paternity. In the AmpFLPs technique, thepolynucleotides may be labeled by using a labeled oligonucleotide PCRprimer, or by utilizing labeled nucleotide triphosphates in the PCR.

Another fragment analysis method is nick translation. Nick translationinvolves a reaction to replace unlabeled nucleotide triphosphates in adouble-stranded DNA molecule with labeled ones. Free 3′-hydroxyl groupsare created within the unlabeled DNA by “nicks” caused bydeoxyribonuclease I (DNAase I) treatment. DNA polymerase I thencatalyzes the addition of a labeled nucleotide to the 3′-hydroxylterminus of the nick. At the same time, the 5′ to 3′-exonucleaseactivity of this enzyme eliminates the nucleotide unit from the5′-phosphoryl terminus of the nick. A new nucleotide with a free 3′-OHgroup is incorporated at the position of the original excisednucleotide, and the nick is shifted along by one nucleotide unit in the3′ direction. This 3′ shift will result in the sequential addition ofnew labeled nucleotides to the DNA with the removal of existingunlabeled nucleotides. The nick-translated polynucleotide is thenanalyzed using a separation process, e.g., electrophoresis.

Another exemplary fragment analysis method is based on variable numberof tandem repeats, or VNTRs. VNTRs are regions of double-stranded DNAthat contain adjacent multiple copies of a particular sequence with thenumber of repeating units being variable. Examples of VNTR loci arepYNZ22, pMCT118, and Apo B. A subset of VNTR methods are those methodsbased on the detection of microsatellite repeats, or short tandemrepeats (STRs), i.e., tandem repeats of DNA characterized by a short(2–4 bases) repeated sequence. One of the most abundant interspersedrepetitive DNA families in humans is the (dC-dA)n--(dG-dT)n dinucleotiderepeat family (also called the (CA)n dinucleotide repeat family). Thereare thought to be as many as 50,000 to 100,000 (CA)n repeat regions inthe human genome, typically with 15–30 repeats per block Many of theserepeat regions are polymorphic in length and can therefore serve asuseful genetic markers. Preferably, in VNTR or STR methods, label isintroduced into the polynucleotide fragments by using a dye-labeled PCRprimer.

Another exemplary fragment analysis method is DNA sequencing. Ingeneral, DNA sequencing involves an extension/termination reaction of anoligonucleotide primer. Included in the reaction mixture aredeoxynucleoside triphosphates (dNTPs) which are used to extend theprimer. Also included in the reaction mixture is at least onedideoxynucleoside triphosphate (ddNTP) which when incorporated onto theextended primer prevents the further extension of the primer. After theextension reaction has been terminated, the different terminationproducts that are formed are separated and analyzed in order todetermine the positioning of the different nucleosides.

Fluorescent DNA sequencing may generally be divided into two categories,“dye primer sequencing” and “dye terminator sequencing”. In dye primersequencing, a fluorescent dye is incorporated onto the primer beingextended. Four separate extension/termination reactions are then run inparallel, each extension reaction containing a differentdideoxynucleoside triphosphate (ddNTP) to terminate the extensionreaction. After termination, the reaction products are separated by gelelectrophoresis and analyzed. See, for example, Ansorge et al., NucleicAcids Res. 15 4593–4602 (1987).

In one variation of dye primer sequencing, different primers are used inthe four separate extension/termination reactions, each primercontaining a different spectrally resolvable dye. After termination, thereaction products from the four extension/termination reactions arepooled, electrophoretically separated, and detected in a single lane.See, for example, Smith et al., Nature 321 674–679 (1986). Thus, in thisvariation of dye primer sequencing, by using primers containing a set ofspectrally resolvable dyes, products from more than oneextension/termination reactions can be simultaneously detected.

In dye terminator sequencing, a fluorescent dye is attached to each ofthe dideoxynucleoside triphosphates. An extension/termination reactionis then conducted where a primer is extended using deoxynucleosidetriphosphates until the labeled dideoxynucleoside triphosphate isincorporated into the extended primer to prevent further extension ofthe primer. Once terminated, the reaction products for eachdideoxynucleoside triphosphate are separated and detected. In oneembodiment, separate extension/termination reactions are conducted foreach of the four dideoxynucleoside triphosphates. In another embodiment,a single extension/termination reaction is conducted which contains thefour dideoxynucleoside triphosphates, each labeled with a different,spectrally resolvable fluorescent dye.

Thus according to one aspect of the invention, a method is provided forconducting dye primer sequencing using one or more oligonucleotidereagents of the present invention. According to this method, a mixtureof extended labeled primers are formed by hybridizing a nucleic acidsequence with a fluorescently labeled oligonucleotide primer in thepresence of deoxynucleoside triphosphates, at least onedideoxynucleoside triphosphate and a DNA polymerase. The fluorescentlylabeled oligonucleotide primer includes an oligonucleotide sequencecomplementary to a portion of the nucleic acid sequence being sequenced,and an energy transfer fluorescent dye attached to the oligonucleotide.

According to the method, the DNA polymerase extends the primer with thedeoxynucleoside triphosphates until a dideoxynucleoside triphosphate isincorporated which terminates extension of the primer. Aftertermination, the mixture of extended primers are separated. The sequenceof the nucleic acid sequence is then determined by fluorescentlydetecting the mixture of extended primers formed.

In a further embodiment of this method, four dye primer sequencingreactions are run, each primer sequencing reaction including a differentfluorescently labeled oligonucleotide primer and a differentdideoxynucleoside triphosphate (ddATP, ddCTP, ddGTP and ddTTP). Afterthe four dye primer sequencing reactions are run, the resulting mixturesof extended primers may be pooled. The mixture of extended primers maythen be separated, for example by electrophoresis and the fluorescentsignal from each of the four different fluorescently labeledoligonucleotide primers detected in order to determine the sequence ofthe nucleic acid sequence.

According to a further aspect of the invention, a method is provided forconducting dye terminator sequencing using one or more dideoxynucleosidetriphosphates labeled with an energy transfer dye of the presentinvention. According to this method, a mixture of extended primers areformed by hybridizing a nucleic acid sequence with an oligonucleotideprimer in the presence of deoxynucleoside triphosphates, at least onefluorescently labeled dideoxynucleotide triphosphate and a DNApolymerase. The fluorescently labeled dideoxynucleotide triphosphateincludes a dideoxynucleoside triphosphate labeled with an energytransfer fluorescent dye of the present invention.

According to this method, the DNA polymerase extends the primer with thedeoxynucleoside triphosphates until a fluorescently labeleddideoxynucleoside triphosphate is incorporated into the extended primer.After termination, the mixture of extended primers are separated. Thesequence of the nucleic acid sequence is then determined by detectingthe fluorescently labeled dideoxynucleoside attached to the extendedprimer.

In a further embodiment of this method, the step of forming a mixture ofextended primers includes hybridizing the nucleic acid sequence withfour different fluorescently labeled dideoxynucleoside triphosphates,i.e., a fluorescently labeled dideoxycytosine triphosphate, afluorescently labeled dideoxyadenosine triphosphate, a fluorescentlylabeled dideoxyguanosine triphosphate, and a fluorescently labeleddideoxythymidine triphosphate.

In each of the above-described fragment analysis methods, the labeledoligonucleotides are preferably separated by electrophoretic procedures,e.g. Gould and Matthews, cited above; Rickwood and Hames, Eds., GelElectrophoresis of Nucleic Acids: A Practical Approach, (IRL PressLimited, London, 1981); or Osterman, Methods of Protein and Nucleic AcidResearch, Vol. 1 Springer-Verlag, Berlin, 1984). Preferably the type ofelectrophoretic matrix is crosslinked or uncrosslinked polyacrylamidehaving a concentration (weight to volume) of between about 2–20 weightpercent. More preferably, the polyacrylamide concentration is betweenabout 4–8 percent. Preferably in the context of DNA sequencing inparticular, the electrophoresis matrix includes a strand separating, ordenaturing, agent, e.g., urea, formamide, and the like. Detailedprocedures for constructing such matrices are given by Maniatis et al.,“Fractionation of Low Molecular Weight DNA and RNA in PolyacrylamideGels Containing 98% Formamide or 7M Urea,” in Methods in Enzymology, 65299–305 (1980); Maniatis et al., “Chain Length Determination of SmallDouble- and Single-Stranded DNA Molecules by Polyacrylamide GelElectrophoresis,” Biochemistry, 14 3787–3794 (1975); Maniatis et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory,New York, 1982), pgs. 179–185; and ABI PRISM™ 377 DNA Sequencer User'sManual, Rev. A, January 1995, Chapter 2 (p/n 903433, The Perkin-ElmerCorporation, Foster City, Calif.), each of which are incorporated byreference. The optimal polymer concentration, pH, temperature,concentration of denaturing agent, etc. employed in a particularseparation depends on many factors, including the size range of thenucleic acids to be separated, their base compositions, whether they aresingle stranded or double stranded, and the nature of the classes forwhich information is sought by electrophoresis. Accordingly applicationof the invention may require standard preliminary testing to optimizeconditions for particular separations. By way of example,oligonucleotides having sizes in the range of between about 20–300 baseshave been separated and detected in accordance with the invention in thefollowing matrix: 6 percent polyacrylamide made from 19 parts to 1 partacrylamide to bis-acrylamide, formed in a Tris-borate EDTA buffer at pH8.3.

After electrophoretic separation, the dye-oligonucleotide conjugates aredetected by measuring the fluorescence emission from the dye labeledpolynucleotides. To perform such detection, the labeled polynucleotidesare illuminated by standard light sources, e.g. high intensity mercuryvapor lamps, lasers, or the like. Previously, fluorescein andrhodamine—based dyes and fluorescein-linked energy transfer dyes havebeen used which are excited at a wavelength between 488 and 550 nm.However, the donor dyes used in the energy transfer dyes of the presentinvention typically have absorption maxima below 450 nm and thus may beexcited at shorter wavelengths, preferably between 250 and 450 nm.

IV. Detection Methods Using Shorter Wavelength Light Sources

The present invention also relates to detection methods, such as thedetection methods described above in Section III, in which a shorterwavelength light source is used, preferably a light source emittinglight between 250 and 450 nm. As noted above, several of the energytransfer dyes of the present invention have the feature of having adonor dye with an emission maxima between about 250 and 450 nm and anacceptor dye which has an emission maxima at a wavelength greater thanabout 500 nm. As a result, these dyes enable these shorter wavelengthlight sources to be used. Accordingly, the present invention relates tomethods for using these shorter wavelength light sources. It is notedthat the use of these shorter wavelength light sources in detectionmethods, such as the ones described in Section III, is not intended tobe limited to the energy transfer dyes of the present invention butrather are intended to encompass the use of any energy transfer dyewhich can be excited using light having a wavelength between 250 and 450nm

V. Kits Incorporating the Energy Transfer Dyes

The present invention also relates to kits having combinations of energytransfer dyes and/or reagents. In one embodiment, the kit includes atleast two spectrally resolvable energy transfer dyes according to thepresent invention. In this kit, the energy transfer dyes preferablyinclude the same donor dye so that a single light source is needed toexcite the dyes.

In another embodiment, the kit includes dideoxycytosine triphosphate,dideoxyadenosine triphosphate, dideoxyguanosine triphosphate, anddideoxythymidine triphosphate, each dideoxynucleotide triphosphatelabeled with an energy transfer dye according to the present invention.In one embodiment, each energy transfer dye is spectrally resolvablefrom the other energy transfer dyes attached to the otherdideoxynucleotide triphosphates. In this kit, the energy transfer dyespreferably include the same first xanthene dye.

In yet another embodiment, the kit includes at least twooligonucleotides, each oligonucleotide including an energy transfer dyeaccording to the present invention. In one embodiment, eacholigonucleotide contains an energy transfer dye which is spectrallyresolvable from the energy transfer dyes attached to the otheroligonucleotides. In another embodiment, the kit includes at least fouroligonucleotides which each contain a spectrally resolvable energytransfer dye.

The energy transfer dyes and their use in DNA sequencing is illustratedby the following examples. Further objectives and advantages other thanthose set forth above become apparent from the examples.

EXAMPLES

1. Method of Synthesis of DYE104

A solution of 5CF-B (8 mg in 0.45 mL dimethylformamide (DMF), 20 μL) wasadded to a solution of the succimidyl ester of coumarin DYE114 (20 μL ofa 5 mg/200 μL DMF solution). Diisopropylethylamine (5 μL) was added.After 5 min, 200 μL of 5% HCl was added. The mixture was centrifuged.The solid was dissolved in bicarbonate solution and purified byreverse-phase HPLC. The synthesis scheme of DYE104 is illustrated inFIG. 9.

2. Method of Synthesis of DYE106

A solution of 5CF-B (8 mg in 0.45 mL dimethylformamide (DMF), 20 μL) wasadded to a solution of the succimidyl ester of coumarin DYE116 (20 μL ofa 5 mg/200 μL DMF solution). Diisopropylethylamine (5 μL) was added.After 5 min, 200 μL of 5% HCl was added. The mixture was centrifuged.The solid was dissolved in bicarbonate solution and purified byreverse-phase HPLC. The synthesis scheme of DYE106 is illustrated inFIG. 10.

2. Method of Synthesis of DYE108

A solution of 5CF-B (8 mg in 0.45 mL dimethylformamide (DMF), 20 μL) wasadded to a solution (20 μL of a 5 mg/200 μL DMF solution) of DYE110,trisulfopyrene acetyl azide (or Cascade Blue acetyl azide, MolecularProbes). Diisopropylethylamine (5 μL) was added. After 5 min, 200 μL of5% HCl was added. The mixture was centrifuged. The solid was dissolvedin bicarbonate solution and purified by reverse-phase HPLC. Thesynthesis scheme of DYE108 is illustrated in FIG. 11.

3. Comparison of Fluorescence Emission Spectra of 5CF-B-Conjugates

The following example compares the fluorescence emission spectra of aseries of energy transfer dyes according to the present invention. Dyesolutions of 5CF-B, DYE102, DYE104, DYE106, and DYE108 were measured inTris-EDTA.

The structures of these dyes are illustrated in FIG. 1. FIG. 12 providesa graph of the relative fluorescence emission of each of these dyes whenexcited at 365 nm. FIG. 12 also show the emission maxima of theindividual dye components. As shown in FIG. 12, the emissions of thedonor dyes do not overlap with the absorbance of the acceptor dye. Thebest conjugate, DYE108, is more than 10-fold brighter than 5CF-B alone.

Table 1 shows the relative spectral data and relative quantum yields of5CF-B conjugates. As can be seen from Table 1, the quantum yields arehigh and the energy transfer is practically quantitative, as observed bythe lack of emission of the donor dyes. Coumarin based dyes DYE104 andDYE102 and pyrene based dye DYE108 display high quantum yieldsindicating that the acceptor is able to absorb substantially all of theenergy emitted by the donor dye. In contrast, the DYE106 (coumarin)displays poor quantum yield and inefficient energy transfer.

TABLE 1 Ex/Em Maxima of Quantum Yield of Conjugate 5CF-B conjugateIndividual Dyes (nm) Relative to 5CF-B 5CF-B 495/523 1.00 DYE106 376/4680.17 DYE104 328/386 0.93 DYE108 396/410 0.91 DYE102 362/459 0.874. Method of Synthesis of Pyrenetrisulfonate-Rhodamine Dye (DYE120)

D-Rox succinimidyl ester (3 mg), 1,4-cyclohexanediamine (7 mg), DMF (100μL) and diisopropylethylamine (10 μL) were combined. After 5 min ethylether was added. The mixture was centrifuged and decanted. The residuewas dissolved in methanol and an aliquot was purified by reverse-phaseHPLC to separate the d-Rox-acid from the the d-Rox-cyclohexanediamineadduct. The purified adduct was concentrated to dryness and dissolved in10 μL DMF.

A solution of DYE110, Cascade Blue acetyl azide, was made (MolecularProbes, 8 mg/100 μL DMF). To 5 μL of the Dye110 solution was added thed-Rox-cyclohexanediamine adduct and 2 μL diisopropylamine. The mixturewas purified by reverse-phase HPLC. The synthesis scheme ofpyrenetrisulfonate-d-Rox dye (DYE120) is illustrated in FIG. 13.

Normalized excitation and emission spectra of thepyrenetrisulfonate-d-Rox adduct (DYE120) are shown in FIG. 14. Verylittle pyrenetrisulfonate emission (410 nm) was observed. The excitationspectra showed a peak at 400 nm that was 50% of the maximum peak at 600nm.

While the present invention is disclosed by reference to the preferredembodiments and examples detailed above, it is to be understood thatthese examples are intended in an illustrative rather than limitingsense, as it is contemplated that modifications will readily occur tothose skilled in the art, which modifications will be within the spiritof the invention and the scope of the appended claims. With regard toall of the molecular structures provided herein, it is intended thatthese molecular structures encompass not only the exact electronicstructure presented, but also include all resonant structures andprotonation states thereof.

1. A fluorescently labeled reagent comprising: a reagent selected trainthe group consisting of a nucleoside, a nucleotide, an oligonucleotideand an oligonucleotide analog; and an energy transfer fluorescent labelattached thereto, the energy transfer fluorescent label comprising: adonor dye having an absorption maximum at a wavelength between 250 and400 nm; and an acceptor dye capable of absorbing excitation energyemitted by the donor dye and emitting light in response, the acceptordye having an emission maximum of greater than about 500 nm, wherebyexcitation of the donor dye with light at said absorption maximum causessaid acceptor dye to emit light at said emission maximum.
 2. Thefluorescently labeled reagent of claim 1 wherein the absorbance maximumof the donor dye is between about 300 and 400 nm.
 3. The fluorescentlylabeled reagent of claim 1 wherein the absorbance maximum of the donordye is between about 350 and 400nm.
 4. The fluorescently labeled reagentof claim 1 wherein the emission maximum of the acceptor dye is greaterthan about 550 nm.
 5. The fluorescently labeled reagent of claim 1wherein the emission maximum of the acceptor dye is between about 500and 700 nm.
 6. The fluorescently labeled reagent of claim 1 wherein theemission maximum of the acceptor dye is at least about 150 nm greaterthan the absorption maximum of the donor dye.
 7. The fluorescentlylabeled reagent of claim 1 wherein the acceptor dye is a member of aclass of dyes selected from the group consisting of fluorescein,rhodamine, asymmetric benzoxanthene, xanthene, cyanine, phthalocyanineand squaraine dyes.
 8. The fluorescently labeled reagent of claim 1wherein the acceptor dye is selected from the group consisting of4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes, rhodamine,4,7dichlororhdamine dyes, carboxyrhodamines, N,N,N′,N′-tetramethylcarboxyrhodamines, carboxy R110, carboxy R6G, carboxy-X-rhodamines andCy5.
 9. The fluorescently labeled reagent of claim 1 wherein theacceptor dye is selected from the group consisting of R110, RG6, TAMRAand ROX.
 10. The fluorescently labeled reagent of claim 1 that furthercomprises a linker linking the donor dye to the acceptor dye.
 11. Thefluorescently labeled reagent of claim 10 wherein the linker absorbssubstantially all of the excitation energy emitted by the donor dye andcomprises a functional group selected from the group consisting of analkene, a diene, an alkyne, a five or six membered ring having at leastone unsaturated bond and a fused ring structure.
 12. The fluorescentlylabeled reagent of claim 11 wherein the functional group is selectedfrom the group consisting of cyclopenetene, cyclohexene,cyclopentadiene, cyclohexadiene, furan, thifuran, pyrrole, isopyrole,isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine,pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran,thionaphthene, indole and naphthalene.
 13. The fluorescently labeledreagent of claim 1 wherein the reagent is a nucleoside.
 14. Thefluorescently labeled reagent of claim 1 wherein the reagent is anucleotide.
 15. The fluorescently labeled reagent of claim 14 whereinthe nucleotide is a 2′-deoxyribonucleotide.
 16. The fluorescentlylabeled reagent of claim 1 wherein the reagent is an oligonucleotide.17. The fluorescently labeled reagent of claim 16 wherein theoligonucleotide has a 3′ end which is not extendable using a polymerase.18. A fluorescently labeled reagent comprising: a reagent selected fromthe group consisting of a nucleoside, a nucleoside monophosphate, anucleoside diphosphate, a nucleoside triphosphate, an oligonucleotideand an oligonucleotide analog; and an energy transfer fluorescent dyeattached to the reagent, the energy transfer fluorescent dye beingselected from the group consisting of DYE102, DYE104, DYE106, DYE108,DYE118, and DYE120.
 19. The fluorescently labeled reagent of claim 13wherein the nucleoside is a 2′-deoxyribonucleoside.
 20. Thefluorescently labeled reagent of claim 19 wherein the2′-dideoxyribonucleoside is selected from the group consisting of2′-dideoxyriboadenosine, 2′-dideoxyribocytidine, 2′-dideoxyribognanosineand 2′-dideoxyribothymidine.
 21. The fluorescently labeled reagent ofclaim 13 wherein the nucleotide is a 2′,3′-dideoxyribonucleotide. 22.The fluorescently labeled reagent of claim 21 wherein the2′,3′-deoxyribonucleotide is selected from the group consisting of a2′,3′-deoxyriboadenosine, 2′,3′-dideoxyribognanosine and2′,3′-dideoxyribothymidine.
 23. The fluorescently labeled reagent ofclaim 15 wherein the 2′-deoxyribonucleotide is selected from the groupconsisting of a 2′-deoxyribonucleotide-5′-monophosphate, a2′-deoxyribonucleotide-5′-diphosphate and a2′-deoxyribonucletide-5′-triphosphate.
 24. The fluorescently labeledreagent of claim 15 wherein the 2′-dideoxyribonucleotide is selectedfrom the group consisting of 2′-dideoxyribocytidine-5′-triphosphate,2′-dideoxyriboguanosine-5′-triphosphate,2′,3′-dideoxyriboguanosine5′-triphosphate and2′,3′-dideoxyribothymidine-52′deoxyribothymidine-5′-triphosphate. 25.The fluorescently labeled reagent of claim 14 wherein the nucleoside isa 2′,3′-dideoxyribonucleoside.
 26. The fluorescently labeled reagent ofclaim 25 wherein the 2′,3′-dideoxyribonucleotide is selected from thegroup consisting of a 2′,3′-dideoxyribonucleotide-5′-monophosphate, a2′,3′-dideoxyribonucleotide-5′-diphosphate and a2′,3′-dideoxyribonucleotide-5′-triphosphate.
 27. The fluorescentlylabeled reagent of claim 25 wherein the 2′,3′-deoxyribonucleotide isselected from the group consisting of2′,3′-deoxyriboadenosine-5′,triphosphate,2′,3′-dideoxyribocytidine-5′-triphosphate,2′,3+-deoxyriboguanosine-5′-trophosphate and2′-deoxyribothymidine-5′-triphosphate.
 28. The fluorescently labeledreagent of claim 1 wherein the donor dye comprises a coumarin ring, apyrene ring or a sulfopyrene ring.